Composition and method for preventing or treating a virus infection

ABSTRACT

The present invention provides multiple antigenic agents compositions and the use thereof to prevent or treat viral infections.

This application claims benefit under 35 U.S.C. 371 fromPCT/US2006/008851, filed Mar. 13, 2006, which is a Continuation-in-PartApplication of U.S. patent application Ser. No. 11/093,107, filed Mar.29, 2005, now U.S. Pat. No. 7,354,589.

BACKGROUND OF THE INVENTION

Three types of transmembrane proteins are expressed in the membrane ofinfluenza type A virions and virus-infected cells. The hemagglutinin andneuraminidase are glycoproteins with large ectodomains of ˜510 and ˜420amino acids, respectively. Hemagglutinin is assembled as homotrimers andneuraminidase as homotetramers forming a dense layer of 13-14 nm long,rod-shaped surface projections on the viral membrane and at cellularsites of virus maturation. Current influenza virus vaccines aim atinducing a strong antibody response to these glycoproteins, particularlythe hemagglutinin, as such antibodies are well-known to be highlyprotective against infection. The problem is that influenza type A virushas a high propensity for changing the determinants recognized by theseprotective antibodies, which necessitates repetitive vaccination withupdated vaccine strains that reflect these antigenic changes. Bycontrast, the third viral transmembrane protein, matrix protein 2 (M2),contains an ectodomain (M2e) that is conserved amongst human influenzavirus strains. Broad protective immunity against influenza type A virusinfection using M2 has been investigated (Slepushkin, et al. (1995)Vaccine 13:1399-1402; Frace, et al. (1999) Vaccine 17:2237-44; Neirynck,et al. (1999) Nature Med. 5:1157-63; Okuda, et al. (2001) Vaccine19:3681-91).

M2 is a 97 amino acid long transmembrane protein of influenza type Avirus (Lamb, et al. (1981) Proc. Natl. Acad. Sci. USA 78:4170-4174;Lamb, et al. (1985) Cell 40:627-633). The mature protein formshomotetramers (Holsinger and Lamb (1991) Virology 183:32-43; Sugrue andHay (1991) Virology 180:617-624) that have pH-inducible ion channelactivity (Pinto, et al. (1992) Cell 69:517-528; Sugrue and Hay (1991)supra). M2-tetramers are expressed at high density in the plasmamembrane of infected cells but are relatively excluded from sites ofvirus maturation and therefore are incorporated only at a low frequencyinto the membrane of mature virus particles (Takeda, et al. (2003) Proc.Natl. Acad. Sci. USA 100:14610-14617; Zebedee and Lamb (1988) J. Virol.62:2762-2772). The sequence of the 24 amino acid long ectodomain of M2(M2e) has remained conserved amongst human epidemic virus strains(Macken, et al. (2001) In Options for the Control of Influenza. IV.Osterhaus, et al. (ed.), p. 103-106. Elsevier Science, Amsterdam). Themajority of human epidemic strains isolated since 1918 share the sameM2e protein sequence. Further, several studies in mice have shown thatM2e-specific antibodies restrict influenza virus replication and reducemorbidity and mortality (Fan, et al. (2004) Vaccine 22:2993-3003; Liu,et al. (2004) Immunol. Lett. 93:131-136; Mozdzanowska, et al. (2003) J.Virol. 77:8322-8328; Neirynck, et al. (1999) supra; Treanor, et al.(1990) J. Virol. 64:1375-1377). Moreover, in ferrets, the animal modelconsidered most prognostic for human influenza, protective activity ofM2e-specific immunity has been demonstrated (Fan, et al. (2004) supra)and sera from rhesus monkeys immunized with a M2e-carrier conjugate havebeen shown to exhibit protective activity upon transfer into mice (Fan,et al. (2004) supra). Thus, with the exception of a study in pigs, whichindicated that M2e-specific immunity may enhance rather than amelioratedisease (Heinen, et al. (2002) J. Gen. Virol. 83:1851-1859), evidencefrom animal models shows that M2e-specific immunity is capable ofproviding a significant level of protection that is directed against aremarkably conserved viral target.

The low degree of structural variation in M2e is certainly in partattributable to constraints resulting from its genetic relation to M1,the most conserved protein of the virus (Ito, et al. (1991) J. Virol.65:5491-5498). M2 is encoded by a spliced RNA of the viral gene segment7, which codes also for M1 (Lamb, et al. (1985) supra). The splicingevent removes most of the nucleotides that code for M1 (nt 27-714)except the 26 most 5′ and 42 most 3′ nucleotides (Lamb, et al. (1985)supra). Thus, nucleotides 1-68 of M2 which encode essentially the entireM2e are bicistronic, from 1-26 in the same and from 27-68 in a differentreading frame. This genetic relation between M2e and M1 can be expectedto substantially restrict the degree of variability in M2e. Anadditional factor that may contribute to the low degree of change seenin M2e amongst human influenza virus strains could be the absence ofM2e-specific antibodies and thus pressure for change. A small study of17 paired human sera obtained during the acute and convalescent phase ofnatural infection found that M2-specific antibodies were absent fromacute sera and became detectable in only six of the convalescents(Black, et al. (1993) J. Gen. Virol. 74 (Pt 1):143-146). This was incontrast to nucleoprotein-specific antibody titers, which increased in15 of 17 convalescent sera, thus confirming recent influenza infectionof the donors (Black, et al. (1993) supra). Another study found nodifference in M2e-specific antibody titers in two larger groups ofunpaired sera, one positive and the other negative for virus-specificantibodies (Liu, et al. (2003) FEMS Immunol. Med. Microbiol.35:141-146). These data suggest that while infection in humans canresult in a measurable antibody response to M2, the response is notgenerated consistently and is small and of short duration. Similarobservations have been made in the mouse model where two repetitiveinfections with virus strains that shared the same M2e induced only lowtiters of M2e-specific antibodies (Mozdzanowska, et al. (2003) Vaccine21:2616-2626). Since M2 is a minor component (<0.5%) of purified virus(Zebedee and Lamb (1988) J. Virol. 62:2762-2772), inactivated influenzavirus vaccines presently being used would not be expected to induceM2e-specific immunity either.

M2e-specific monoclonal antibody 14C2 does not prevent virus infectionin vitro but reduces virus yield and plaque size when incorporated intothe culture medium or agar overlay (Zebedee and Lamb (1988) supra;Hughey, et al. (1995) Virology 212:411-21). Not all M2e-specificantibodies display this activity (Hughey, et al. (1995) supra) and notall virus strains are susceptible to it (Zebedee and Lamb (1988) supra).In vivo, passive monoclonal antibody 14C2 similarly decreases virusgrowth (Treanor, et al. (1990) J. Virol. 64:1375-7) and is effectivealso against PR8 (Mozdzanowska, et al. (1999) Virology 254:138-46),which is not susceptible to antibody-mediated growth restriction invitro (Zebedee and Lamb (1988) supra; Mozdzanowska, et al. (1999)supra), indicating that antibody-mediated virus growth-inhibition occursthrough distinct mechanisms in vitro and in vivo.

It has now been found that a multiple antigenic agent containing M2elinked to helper T cell determinants is an effective vaccine forinducing virus protection. M2e-MAAs together with cholera toxin (CT) anda synthetic oligodeoxynucleotide (ODN) with a stimulatory CpG motifinduces strong M2e-specific antibody titers in serum of mice and resultsin significant protection against influenza virus challenge.

SUMMARY OF THE INVENTION

The present invention is a multiple antigenic agent (MAA) of thestructure:

(SEQ ID NO:1) wherein, R₁ is 0 to 2 amino acid residues comprising Cysor Gly or a nucleic acid sequence; m is at least 1; n is at least 1;Xaa₁ is 0 to 1 amino acid residue comprising

or Gly; R₂, R₃, and R₄ may independently be a B cell determinant, a Tcell determinant, or a targeting molecule; and R₅ is an amino acid,peptide, or nucleic acid sequence. In one embodiment, when m is greaterthan 1, each R₂ can independently be a B cell determinant, a T celldeterminant, or a targeting molecule; and when n is greater than 1, eachR₃ can independently be a B cell determinant, a T cell determinant, or atargeting molecule. In a particular embodiment, the B cell determinantis the ectodomain of matrix protein 2, or a fragment or homolog thereof.In another embodiment, a Cys residue located at the N-terminus of afirst MAA is covalently linked via a disulfide bond to a second Cysresidue at the N-terminus of a second MAA of Formula I to produce an MAAdimer of Formula I.

The present invention is also a composition containing an MAA and apharmaceutically acceptable carrier. In one embodiment, the compositioncontaining the MAA and the pharmaceutically acceptable carrier mayfurther contain an adjuvant. Such compositions are useful for preventingor treating a viral infection. Accordingly, a method for preventing ortreating a viral infection is provided involving administering to asusceptible subject or one exhibiting signs or symptoms of viralinfection an effective amount of a composition of the invention toprevent or treat the signs or symptoms of a viral infection. Inparticular embodiment, the viral infection is influenza type A virus.

These and other aspects of the present invention are set forth in moredetail in the following description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effects of M2e-MAA dose on M2e antibody titer. BALB/cmice were immunized i.n. with the indicated doses of an MAA containingfour M2e24 B cell determinants in adjuvant. M2e-specific serum antibodytiters were determined three weeks after each immunization. Mean valuesfrom two independent experiments are shown.

FIG. 2 shows specificity of the response induced by immunization with(4)M2e-MAA. FIG. 2A, groups of four mice were immunized with the samedose of (4)M2e25-MAA in adjuvant by the i.n., s.c. or i.p. route. Seraobtained after first and second booster immunizations were tested forspecificity by ELISA for antibody titer at 21 days after the firstbooster immunization (b21), 14 days after the second boosterimmunization (2b14), and 90 days after the second booster immunization(2b90). Binding to each immunosorbent was quantified by comparison tothe binding seen with purified anti-M2e monoclonal antibody. FIG. 2B,groups of four mice were immunized by the i.n. route with (4)M2e24-MAPor (4)M2e15-MAP in adjuvant and sera tested for specificity as in FIG.2B.

DETAILED DESCRIPTION OF THE INVENTION

It has now been found that animals, inoculated with multiple antigenicagents (MAAs) containing multiple B cell determinants and T helper cell(Th) determinants, exhibit significant resistance against subsequentchallenge with infectious virus. As defined herein, a multiple antigenicagent is an agent which contains more than one peptide or nucleic acidmoiety which is capable of inducing a specific immune response in ananimal. The B cell determinant induces an antibody response and can alsoinduce a T cell response. The advantage of the MAAs provided herein isthat a multitude of antigenic side chains can be attached to the corepeptide, which contains Lys-Gly repeats, thereby enabling presentationof several structurally linked determinants. Furthermore, when a Cysresidue is linked at the N-terminus of the core peptide, two corepeptides can be covalently linked via disulfide bonds to effectivelydouble the number of antigenic side chains and hence improve immuneresponses in mammals. Further, as the MAA provided herein can be readilychemically synthesized, the production of the MAA is highly controlledand contaminants are minimized.

Accordingly, the present invention is an MAA of the structure:

(SEQ ID NO:1), wherein m is at least 1 and n is at least 1. In oneembodiment, the summation of m and n is about 10 to 30. In anotherembodiment the summation of m and n is about 10.

In the MAA of Formula I, the amino acid moiety Xaa₁ is 0 to 1 amino acidresidue, wherein when Xaa₁ is 1 amino acid residue, the amino acid is

or Gly. In particular embodiments, Xaa₁ is Gly.

In the MAA of Formula I, the R₁ moiety is 0 to 2 amino acid residues ora nucleic acid sequence such as an oligodeoxynucleotide (ODN) with astimulatory CpG motif. In one embodiment, the R₁ moiety is 0 amino acidresidues. In another embodiment, the R₁ moiety is 1 amino acid residue.When R₁ is 1 amino acid residue, the amino acid residue can be a Gly orCys. In yet another embodiment, R₁ is the dipeptide Cys-Gly. When theN-terminal amino acid of a first MAA is a Cys-Gly dipeptide, the Cysresidue is desirably covalently linked to a second Cys residue at theN-terminus of a second MAA of Formula I. The disulfide linkage betweenthe first and second cysteine residues generates a covalently linked MAAdimer of Formula I. It is contemplated that the peptides of the dimercan be identical or differ in the number of Lys-Gly repeats (i.e., thenumber of m and n), Xaa₁, R₂, R₃, R₄ or R₅.

In the MAA of Formula I, the R₂, R₃ and R₄ substituents attached to theε-amino group of lysine can independently be a B cell determinant, a Tcell determinant, or a targeting molecule. In one embodiment, at leastone B cell determinant and one T-cell determinant are present in the MAAof Formula I. In another embodiment, the MAA of Formula I has at leasttwo B cell determinants. In still a further embodiment, the MAA ofFormula I has at least four B cell determinants. Advantageously, the MAAof Formula I can have various combinations and numbers of B celldeterminants, T cell determinants, or targeting molecules from varioussources (e.g., from different viral families or serotypes). Toillustrate, a monomer MAA of the invention can contain five B celldeterminants, two T cell determinants and two targeting molecule sidechains. Alternatively, a monomer MAA of the invention can, for example,contain eight B cell determinants, one T cell determinant and onetargeting molecule side chain. The combinations are not particularlylimited and can vary with the selected B cell determinant, T celldeterminant or targeting molecule. Further, individual R groups ofLys-Gly repeats of m or n length can vary. For example, if m=3, R₂ ofthe first Lys-Gly repeat can be a B cell determinant, the R₂ of thesecond Lys-Gly repeat can be a T cell determinant and the R₂ of thethird Lys-Gly repeat can be a targeting molecule.

B cell determinants, as used herein, desirably elicit a measurable Bcell response as determined by, for example, production of antibodies tothe native viral protein. B cell determinants which can be used in theMAA of Formula I include those already known in the art as well as anyother antigens such as glycans, polypeptides, or nucleic acids whichelicit a B cell response. In one embodiment, antigens are derived fromenveloped or non-enveloped viruses. In another embodiment, antigens arederived from viruses including, but not limited to, those from thefamily Adenoviridae, Arenaviridae (e.g., Lymphocytic choriomeningitisvirus), Arterivirus (e.g., Equine arteritis virus), Astroviridae (Humanastrovirus 1), Birnaviridae (e.g., Infectious pancreatic necrosis virus,Infectious bursal disease virus), Bunyaviridae (e.g., Californiaencephalitis virus Group), Caliciviridae (e.g., Caliciviruses),Coronaviridae (e.g., Human coronaviruses 299E and OC43), Deltavirus(e.g., Hepatitis delta virus), Filoviridae (e.g., Marburg virus, Ebolavirus Zaire), Flaviviridae (e.g., Yellow fever virus group, Hepatitis Cvirus), Hepadnaviridae (e.g., Hepatitis B virus), Herpesviridae (e.g.,Epstein-Bar virus, Simplexvirus, Varicellovirus, Cytomegalovirus,Roseolovirus, Lymphocryptovirus, Rhadinovirus), Orthomyxoviridae (e.g.,Influenzavirus A, B, and C), Papovaviridae (e.g., Papillomavirus),Paramyxoviridae (e.g., Paramyxovirus such as human parainfluenza virus1, Morbillivirus such as Measles virus, Rubulavirus such as Mumps virus,Pneumovirus such as Human respiratory syncytial virus), Picornaviridae(e.g., Rhinovirus such as Human rhinovirus 1A, Hepatovirus such Humanhepatitis A virus, Human poliovirus, Cardiovirus such asEncephalomyocarditis virus, Aphthovirus such as Foot-and-mouth diseasevirus O, Coxsackie virus), Poxyiridae (e.g., orthopoxvirus such asVariola virus), Reoviridae (e.g., Rotavirus such as Groups A-Frotaviruses), Retroviridae (Primate lentivirus group such as humanimmunodeficiency virus 1 and 2), Rhabdoviridae (e.g., rabies virus) andTogaviridae (e.g., Rubivirus such as Rubella virus).

An exemplary B cell determinant which can be used in the MAA of FormulaI includes, but is not limited to, the ectodomain of M2, or a fragmentor homolog thereof. In one embodiment, the B cell determinant of the MAAof Formula I is M2e derived from influenza type A set forth herein asSEQ ID NO:11. In a particular embodiment, the B cell determinant of theMAA of Formula I is an M2e fragment represented by SEQ ID NO:12. Instill further embodiments, the B cell determinant of the MAA of FormulaI is a homolog of M2e derived from viral transmembrane proteins such asNB of influenza type B virus or CM2 of influenza type C virus.

T cell determinants are intended to include both Th and cytotoxic T celldeterminants. Elicitation of T cell responses can be detected, forexample, as exemplified herein or by measuring the production ofcytokines, e.g., IFN-gamma, IL-2, IL-4, IL-5, or IL-10. Exemplary T celldeterminants which can be used in the MAA of Formula I include, but arenot limited to, hemagglutinin T cell determinants and T celldeterminants restricted to human MHC class II proteins, preferably to abroad range of haplotypes.

Targeting molecules covalently linked to the antigen as the R₂, R₃, orR₄ moieties are, in general, carbohydrates, lipids, peptides oroligonucleotides which deliver the antigen to the desired site.Targeting molecules which can be incorporated into the MAAs of thepresent invention include, but are not limited to, cholera toxin, ODNswith stimulatory CpG motifs (see, e.g., Shirota, et al. (2001) J.Immunol. 167(1):66-74) and endogenous human immunomodulators, such asIL-2, IL-12, and GM-CSF.

The R₅ moiety of the MAA of Formula I is not particularly limited andcan be any amino acid (e.g., β-linked alanine), peptide, or nucleic acidsequence such as an ODN with a stimulatory CpG motif.

The MAA of Formula I can be prepared in accordance with the methodsexemplified herein or any other suitable method of chemicallysynthesizing peptides and peptide conjugates. Wherein nucleic acidsequences are incorporated into Formula I, said nucleic acid sequencescan be attached via spacer or linker molecules such ashydroxy-carboxylic acid.

Examples provided herein disclose various derivatives of the MAA ofFormula I for inducing an immune response in mice (see Table 1) and areintended to illustrate and not limit the scope of the invention. Otheraspects, advantages and modifications within the scope of the inventionwill be apparent to those skilled in the art to which the inventionpertains.

M2e-MAAs were administered to anesthetized mice by the intranasal (i.n.)route in a dose of 50 μL. Primary and booster inocula contained 3 μg ofMAA, 3 μg of phosphorothionated oligodeoxynucleotide (ODN) 1826, whichcontains an immunostimulatory CpG motif, and 0.5 μg of cholera toxin(CT) in phosphate-buffered saline (PBS) and were given at an interval offour to five weeks. Mice inoculated i.n. with ODN and CT alone or withinfectious virus were used as negative and positive controls,respectively. In the latter case, the first infection was with PR8 andthe second with PR8-SEQ14, a variant that differs from PR8 by 14 aminoacid substitutions in hemagglutinin-determinants recognized byprotective monoclonal antibodies and can readily induce an infection inPR8-immune mice.

Ten to thirty days after boost, cells from mediastinal lymph nodes(MedLNs) were tested for their capacity to proliferate in response tofree S1 peptide, hemagglutinin and M2e. Cells from spleen and lymphnodes draining the upper respiratory tract gave smaller responses andwere less extensively studied. The responses of M2e-MAA-immunized miceconsistently exceeded those of adjuvant-primed control mice. Only two ofthe data sets exceeded the response of the control mice on a statisticalbasis (paired t test, p≧0.05). However, as a group, M2e-MAA-immunizedmice exhibited significantly greater S1- and hemagglutinin-specificresponses than control mice (unpaired t test, p≦0.05). Thehemagglutinin-specific response of M2e-MAA-immunized mice was similar insize to the one of infection-immunized mice but differed in finespecificity in that S1-specific Th were detected in M2e-MAA-immunizedbut not infection-immunized mice. The mannosylated MAA was not superiorto non-mannosylated MAAs in inducing a S1-specific Th response in vivo,in marked in contrast to its greater stimulatory potency in vitro.Further, M2e-MAA-immunized but not infection-immunized mice containedM2e-specific proliferative T cells, indicating that M2e itself containsat least one H2^(d)-restricted Th determinant.

There was no evidence of induction of MHC class I-restricted cytotoxicmemory T (Tc) responses by M2e-MAAs, which is consistent with theabsence of a characteristic K^(d)-restriction, D^(d)-restriction orL^(d)-restriction motif in M2e (Engelhard (1994) Curr. Opin. Immunol.6:13-23; Corr, et al. (1993) J. Exp. Med. 178:1877-92). Memory Tc werereadily detectable in infection-immunized mice.

M2e-specific serum antibody titers were measured by ELISA on solid phaseimmunoadsorbents of (1)M2e24-MAA and JAP-MDCK cell monolayers. Eachassay was standardized and quantified by concomitant titration ofpurified M2e-specific monoclonal antibody 14C2 and antibody titers intest samples were defined as equivalent μg M2e-specific antibody permilliliter of serum.

Combined data from four independent immunization experiments in whichmice were bled 2 and 4 weeks after priming, 2 and 4-5 weeks after secondand 2 and 5 weeks after the third immunization provided the average andSEM of group titers from the different immunization experiments. Thedata indicated that constructs containing four B cell determinants(e.g., (4)M2e24-MAA) induced a prompter and stronger response than theconstructs containing two B cell determinants (e.g., (2)M2e24-MAA) andthe latter was superior to constructs containing one B cell determinant(e.g., (1)M2e24-MAA). Unexpectedly, and in contrast to the enhancedstimulation of Th cells in vitro, mannosylation decreased the MAA'sability to induce an antibody response in vivo. This held for both theantibody titer measured against a construct containing one M2e24 (i.e.,(1)M2e24-MAA) and JAP-MDCK cells. Immunization with constructscontaining four M2e24 B cell determinants consistently inducedsignificant antibody titers two weeks after the second immunization andsometimes induced a significant response as soon as four weeks afterprimary immunization (in four independent experiments, mean titers of1.0, 2.0, 4.6, and 1035 μg/mL 4 weeks after first immunization). Thefindings indicate that a multimeric presentation of M2e enhanced the Bcell response, by facilitating the cross-linking of membrane Ig onM2e-specific B cells (Bachmann and Zinkernagel (1997) Annu. Rev.Immunol. 15:235-70).

A dose of ˜3 μg of M2e-MAA administered i.n. with adjuvant induced anoptimal response as compared to doses of 0.1, 0.6 and 15 μg. The ˜3 μgdose induced a minor primary response but a substantial secondary andtertiary antibody response (FIG. 1). The tertiary response was moreuniform in terms of titer than the secondary response and was of longduration (>60 weeks). However, a dose as little as 0.6 μg induced asizable secondary antibody response (FIG. 1).

The data further indicated that sera from M2e-MAA-immunized miceconsistently exhibited higher titers against M2e-MAA than againstJAP-MDCK cells. The ratio of M2e-MAA versus JAP-MDCK reactive antibodiesin sera showed an average of 10 and ranged in individual sera from twoto 31. This indicates that the specificity of the antibody responseafter M2e-MAA immunization differed amongst individual mice and that onaverage ˜10% of the M2e-MAA specific antibodies cross-reacted withvirus-induced M2e on infected cells. The residual antibodies may bedirected to Th-determinants, scaffold peptide, determinants on thesynthetic M2e-peptide that are not shared by the virus-inducedM2-tetramer or combinations of these structures.

ELISA against M2e-MAA further indicated that sera from virus-infectedmice contained very low M2e-specific antibody titers. The ELISA againstJAP-MDCK detects antibodies to many viral proteins and was therefore notused to quantify the M2-specific response in infection-immunized mice.The only exception was one group of mice that had been immunized bythree consecutive infections, first by PR8, second by JAP and third byX31 and exhibited a M2e-MAA-specific titer of ˜30 μg/mL, which was inthe same range as the viral M2e-specific antibody titers (versusJAP-MDCK) seen in mice immunized with (4)M2e24-MAA. The data showed that(4)M2e-MAA was more effective than virus infection in inducing aM2e-specific antibody response.

M2e-specific immune effectors were virtually absent from mice immunizedby two consecutive infections. This is unexpected, considering that M2is expressed at high density in the plasma membrane of infected cells(Lamb, et al. (1985) supra; Zebedee and Lamb (1988) supra) and that avast number of epithelial cells become infected in the course of a totalrespiratory tract infection (Yilma, et al. (1979) J. Inf. Disease139:458-64). This finding indicates that the strength of heterosubtypicprotection may be enhanced by concomitant induction of the effectorsinduced by infection and M2e-specific vaccination.

Four to five weeks after the second immunization, mice were challengedwith X31 and virus titer in nasal, tracheal and pulmonary tissuesdetermined three days later. At this time, virus titer is affectedalmost exclusively by innate and extant memory defense activities asnaïve SCID and immunocompetent mice show no difference in virus titer atthis time point after challenge. Mice immunized with MAAs that containeda single M2e, with or without mannose, exhibited no significantresistance (ns, p>0.01 by student t test) to virus replication in nasaland pulmonary tissues but showed reduced virus replication in thetrachea compared to mice primed with adjuvant alone. Conversely, in miceprimed and boosted with (4)M2e24-MAA, the virus titer in nose, tracheaand lung three days after total respiratory tract challenge was reducedon average by 10- to 100-fold compared to mice immunized with adjuvantalone. The protection was of similar strength in nose and trachea asseen in infection-immunized mice but of lesser strength in the lung whenassessed 4 weeks after the boost. However, since infection-inducedprotection is largely memory T cell-mediated and appears to be ofshorter duration than antibody-mediated protection, M2e-MAA-inducedprotection may be of longer duration than infection-induced protection.

Co-administration of (4)M2e24-MAA and infectious virus resulted in aslight increase in protection in nose and trachea but not lung, comparedto mice immunized with (4)M2e24-MAA or infection alone. However, whileinfection increased the fraction of G2a antibodies, it decreased thesize of the total M2e-specific antibody response in serum; serumantibody is known to play an important role in protection in the lungand less so in the nose and trachea.

M2e-specific serum antibody titers were tested in individual mice forcorrelation with virus titers. Antibody titers and nasal and pulmonary,but not tracheal, virus titers correlated inversely in(4)M2e-MAA-immunized mice (correlation coefficient, R², for nose andlung 0.53 and 0.51, respectively, p<0.001). However, a substantialfraction of the correlation was due to the single, outlying mouse thatcontained ˜90 μg anti-M2e antibody per mL of serum. Its exclusionreduced the correlation between antibody and virus titer to aninsignificant value in the nose but not in the lung, where it remainedsignificant (R² 0.41, p=0.002). No correlation was seen betweenM2e-specific antibody and virus titers in trachea and in mice immunizedby infection.

Intranasal immunization with (4)M2e24-MAA in adjuvant induced antibodiesof three main specificities: M2e-specific antibodies that cross-reactedwith native M2e; M2e-peptide-specific antibodies that did not react withnative M2e, and antibodies that were specific for determinants ofM2e-MAAs other than M2e. These responses were assessed in ELISA against(2)M2e24-MAP, which provides a measure for the total response;Cys-M2e24, which provides an estimate for the M2e- and backbone-specificresponse; virus-infected MDCK cells, which measures the antibody titerto native M2e; and Cys-bb, which measures the response to MAA backbone(and assay background). On average, one-third of the response wasdirected to Th determinants and background, two-thirds (66±17%) wasspecific for M2e and approximately one-fifth (17±11%, SD; n=12) fornative M2e. The response to native M2e can be further improved by usingextrapulmonary routes of immunization (FIG. 2A) and M2e-MAAs that haveC-terminal truncations (FIG. 2B).

The isotype composition of the M2e-specific antibody response induced byi.n. immunization with (4)M2e24-MAA was dominated by IgG1, an isotypethat is less protective in the case of M2e-specific antibodies thanIgG2a. Administration of M2e-MAA concomitantly with 100 TCID₅₀ ofinfectious PR8 (or PR8-Seq14) virus resulted in an increase of G2a anddecrease of G1 isotype; however, the total size of the response wasconcomitantly reduced.

These results indicate that i.n. immunization with M2e-MAA in adjuvantprovided significant protection that was almost as strong as theheterosubtypic protection resulting from two consecutive totalrespiratory tract infections. Further, M2e-specific protection can befurther enhanced by increasing antibody titer (i.e., three instead oftwo injections), fraction of native M2e-specific antibody (i.e.,truncation of C-terminal and extrapulmonary routes of immunization) andby biasing the isotype composition to G2a.

Given these results, the present invention is also a method for usingthe MAAs both as therapeutic and prophylactic agents for treating orpreventing viral infections. In general, this will involve administeringan effective amount of one or more MAAs of the present invention in asuitable form to a susceptible subject or one exhibiting signs orsymptoms of viral infection.

As will be appreciated by the skilled artisan, the selection of the Bcell determinant for the MAA of Formula I will be dependent on the viralinfection to be prevented or treated. For example, to prevent or treatan influenza viral infection, the B cell determinant of the MAA ofFormula I should be derived from influenza virus (e.g., M2e). In usingcognate B cell determinants, it is contemplated that the MAA of FormulaI will be effective in generating an immune response against envelopedor non-enveloped viruses including, but not limited to, those from thefamily Adenoviridae, Arenaviridae (e.g., Lymphocytic choriomeningitisvirus), Arterivirus (e.g., Equine arteritis virus), Astroviridae (Humanastrovirus 1), Birnaviridae (e.g., Infectious pancreatic necrosis virus,Infectious bursal disease virus), Bunyaviridae (e.g., Californiaencephalitis virus Group), Caliciviridae (e.g., Caliciviruses),Coronaviridae (e.g., Human coronaviruses 299E and OC43), Deltavirus(e.g., Hepatitis delta virus), Filoviridae (e.g., Marburg virus, Ebolavirus Zaire), Flaviviridae (e.g., Yellow fever virus group, Hepatitis Cvirus), Hepadnaviridae (e.g., Hepatitis B virus), Herpesviridae (e.g.,Epstein-Bar virus, Simplexvirus, Varicellovirus, Cytomegalovirus,Roseolovirus, Lymphocryptovirus, Rhadinovirus), Orthomyxoviridae (e.g.,Influenzavirus A, B, and C), Papovaviridae (e.g., Papillomavirus),Paramyxoviridae (e.g., Paramyxovirus such as human parainfluenza virus1, Morbillivirus such as Measles virus, Rubulavirus such as Mumps virus,Pneumovirus such as Human respiratory syncytial virus), Picornaviridae(e.g., Rhinovirus such as Human rhinovirus 1A, Hepatovirus such Humanhepatitis A virus, Human poliovirus, Cardiovirus such asEncephalomyocarditis virus, Aphthovirus such as Foot-and-mouth diseasevirus O, Coxsackie virus), Poxyiridae (e.g., Orthopoxvirus such asVariola virus), Reoviridae (e.g., Rotavirus such as Groups A-Frotaviruses), Retroviridae (Primate lentivirus group such as humanimmunodeficiency virus 1 and 2), Rhabdoviridae (e.g., rabies virus) andTogaviridae (e.g., Rubivirus such as Rubella virus).

Treatment of individuals having a viral infection involves identifying asubject exhibiting signs or symptoms of a viral infection andadministering to said subject an effective amount of a MAA of Formula Iof the present invention thereby decreasing the signs or symptomsassociated with the viral infection or abbreviating the duration of theviral infection when compared to a subject who has not receivedtreatment. Signs or symptoms of a viral infection are generallydependent on the particular virus and are well-known to the skilledclinician. For example, typical symptoms of viral infection include, butare not limited to high fever, severe aches and pains, headaches, andsore throat. MAAs for treating viral infections can be used oradministered as a mixture, for example in equal amounts, orindividually, provided in sequence, or administered all at once and canbe administered orally, topically or parenterally in amounts sufficientto effect a reduction in the viral infection signs or symptoms. Further,the MAAs of the present invention can be co-administered with otherwell-known antigens, vaccines or adjuvants.

Likewise, active immunization for the prevention or protection against aviral infection involves administering one or more MAAs as a componentof a vaccine. Vaccination can be performed orally, topically orparenterally in amounts sufficient to enable the recipient subject togenerate protective immunity against the virus of interest to preventthe signs or symptoms of viral infection. An amount is said to besufficient to prevent the signs or symptoms of viral infection if thedosage, route of administration, etc. of the MAA are sufficient toinfluence such a response. Responses to MAA administration can bemeasured by analysis of subject's vital signs or monitoring antibodytiter.

An MAA composition suitable for administration is one which is toleratedby a recipient subject. Such MAA compositions can be prepared accordingto known methods of producing formulations, whereby the MAAs arecombined in admixture with a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are provided, for example, inRemington: The Science and Practice of Pharmacy, Alfonso R. Gennaro,editor, 20th ed. Lippingcott Williams & Wilkins: Philadelphia, Pa.,2000. In order to form an MAA composition suitable for administration,such compositions will contain an effective amount of the MAAs togetherwith a suitable amount of a carrier, excipient, or stabilizer which isnontoxic to the cell or mammal being exposed thereto at the dosages andconcentrations employed. In general, formulations will contain a finalconcentration of MAA in the range of 0.2 μg/mL to 2 mg/mL or moredesirably 5 μg/mL to 500 μg/mL. Often the carrier is an aqueous pHbuffered solution. Examples of pharmaceutically acceptable carriersinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

MAAs, or compositions or formulations containing an MAA of the inventioncan further contain adjuvants to enhance a subject's T cell response tothe antigen. Examples of such adjuvants include, but are not limited to,aluminum salts; Incomplete Freund's adjuvant; threonyl and n-butylderivatives of muramyl dipeptide; lipophilic derivatives of muramyltripeptide; monophosphoryl lipid A; 3′-de-O-acetylated monophosphoryllipid A; cholera toxin; phosphorothionated oligodeoxynucleotides withCpG motifs; and adjuvants such as those disclosed in U.S. Pat. No.6,558,670.

Administration of MAAs, or compositions or formulations containing anMAA disclosed herein can be carried out by any suitable means, includingparenteral injection (such as intraperitoneal, subcutaneous, orintramuscular injection), orally, or by topical application of the MAAs(typically carried in a pharmaceutical formulation) to an airwaysurface.

For injection, the carrier will typically be a liquid, such as sterilepyrogen-free water, pyrogen-free phosphate-buffered saline solution,bacteriostatic water, or Cremophor (BASF, Parsippany, N.J.). For othermethods of administration, the carrier can be either solid or liquid.

Topical application of the MAAs to an airway surface can be carried outby intranasal administration (e.g., by use of dropper, swab, or inhalerwhich deposits a pharmaceutical formulation intranasally). Topicalapplication of the MAAs to an airway surface can also be carried out byinhalation administration, such as by creating respirable particles of apharmaceutical formulation (including both solid particles and liquidparticles) containing the MAAs as an aerosol suspension, and thencausing the subject to inhale the respirable particles. Methods andapparatus for administering respirable particles of pharmaceuticalformulations are well-known, and any conventional technique may beemployed.

Oral administration can be in the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10% to 95% of active ingredient, preferably 25% to 700%.Capsules, tablets and pills for oral administration to a subject may beprovided with an enteric coating comprising, for example, copolymers ofmethacrylic acid and methyl methacrylate, cellulose acetate, celluloseacetate phthalate or hydroxypropylmethyl cellulose.

A composition of the invention can be administered in a mannercompatible with the dosage formulation, and in such amount as will beprophylactically and/or therapeutically effective. The quantity to beadministered, which is generally in the range of 0.1 μg to 250 μg of MAAper dose, depends on the subject to be treated, capacity of thesubject's immune system to synthesize antibodies, and the degree ofprotection desired. A preferable range is from about 0.5 μg to about 20μg per dose.

A suitable dose size is about 0.5 mL. Accordingly, a dose forintramuscular injection, for example, would comprise 0.5 mL containing˜5 μg of MAA in admixture with 0.5% adjuvants.

The exact dosage will be determined by the skilled practitioner, inlight of factors related to the subject that requires treatment. Dosageand administration are adjusted to provide sufficient levels of theactive MAA or to maintain the desired effect of preventing or reducingviral signs or symptoms, or reducing severity of the viral infection.Factors which may be taken into account include the severity of thedisease state, general health of the subject, age, weight, and gender ofthe subject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy.

The composition can be given in a single dose schedule, or desirably ina multiple dose schedule. A multiple dose schedule is one in which aprimary course of administration may be with 1-10 separate doses,followed by other doses given at subsequent time intervals required tomaintain and or reinforce the immune response, for example, at 1 to 4months for a second dose, and if needed, a subsequent dose(s) afterseveral months. The dosage regimen will also, at least in part, bedetermined by the need of the individual and be dependent upon thejudgement of the practitioner.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Synthesis of Multiple Antigenic Agent Constructs (MAAs)

The solid-phase synthesis of exemplary MAA constructs of Formula Ipresented in Table 1 was carried out using a combination of threequasi-orthogonally removable amino protecting groups according towell-known methods (Kragol and Otvos (2001) Tetrahedron 57:957-66).

TABLE 1 Valency SEQ ID of M2e Construct NO: 1 (1) M2e24-Man-MAA

2 (1) M2e24-MAA

3 (1) M2e24-MAP

4 2 (2) M2e24-MAA

5 (2) M2e24-MAP

6 4 (4) M2e24-MAA

7 (4) M2e15-MAP

8 Man = mannose; T cell determinant S1 =Ser-Phe-Glu-Arg-Phe-Glu-Ile-Phe-Pro-Lys-Glu (SEQ ID NO: 9); T celldeterminant S2 = His-Asn-Thr-Asn-Gly-Val-Thr-Ala-Ala-Ser-Ser-His-Glu(SEQ ID NO: 10); B cell determinant M2e24 =Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-Pro-Ile-Arg-Asn-Glu-Trp-Gly-Cys-Arg-Ser-Asn-Asp-Ser-Ser-Asp-Pro(SEQ ID NO: 11); and B cell determinant M2e15 =Ser-Leu-Leu-Thr-Glu-Val-Glu-Thr-(Pro/His/Leu)-Ile-Arg-Asn-Glu-Trp-Gly(SEQ ID NO: 12). In the M2e15 construct, the residue in parenthesis iseither proline, histidine or leucine.

The disulfide-linked octameric peptide construct (4)M2e24-MAA carryingfour copies of M2e24 as well as two copies each of helper T celldeterminants S1 and S2, was made via intermolecular disulfide formationfrom free sulfhydryl-bearing cysteine derivatives in solution (Kragol,et al. (2001) Bioorg. Med. Chem. Lett. 11:1417-20).

Peptide constructs for use as immunosorbents in ELISA to measureantibody titers against MAA backbone and M2e24 constructs respectivelyincluded the Cys-backbone construct consisting of:

-   -   Cys-Gly-Lys-Gly-Lys-Gly-Lys-O-Ala        (SEQ ID NO:13), and the Cys-M2e24 construct consisting of:

(SEQ ID NO:14). These constructs were assembled on a continuous flowautomated peptide synthesizer Miligen 9050 using conventional Fmocchemistry (Fields and Noble (1990) Int. J. Pept. Protein Res.35:161-214). Fmoc TENTAGEL™ S RAM resin (Advanced Chem Tech, Louisville,Ky.) with an initial load of 0.3 mmol/g was used. For chain elongation,a four molar excess of the amino acids was activated in situ with HATU.The coupling times ranged from 1 to 2.5 hours according to couplingdifficulties predicted by the Peptide Companion algorithm (Windowchem,Fairfield, Calif.). During the synthesis of peptide constructCys-backbone, the N-terminal amino group of Cys was protected with a Bocgroup while the Lys side chain amino group carried Aloc protection. Theselective removal of the Aloc group by catalytic hydrogenation, followedby simultaneous peptide chain assembly of two M2e24 peptides gave thefully protected peptide construct Cys-M2e24. The peptides were cleavedfrom the resin by trifluoroacetic acid in the presence of 5% thioanisoland 5% water, and purified by RP-HPLC. The purity of the peptides wasconfirmed by RP-HPLC and MALDI mass spectrometry.

Peptide-DNA chimeras are prepared by one of two methods. On methodinvolves co-synthesizing the peptidic or nucleic acid fragments usingconventional Fmoc-chemistry and suitable hydroxy-carboxylic acid linkers(Soukchareun, et al. (1995) Bioconjugate Chemistry 6:43-53). A secondmethod involves chemical ligation using thiol-containing bifunctionalcoupling reagents (Soukchareun, et al. (1998) Bioconjugate Chemistry9:466-475; Stetsenko and Gait (2000) J. Org. Chem. 65:4900-4908).

Example 2 Media and Solutions

ISC-CM consisted of Iscove's Dulbecco's medium (Life Technologies,Gaithersburg, Md.) supplemented with 2-mercaptoethanol at 0.05 mM,transferrin (Sigma, St. Louis, Mo.) at 0.005 mg/ml, glutamine (JRHBiosciences, Lenexa, Kans.) at 2 mM and gentamicin (Mediatech, Herndon,Va.) at 0.05 mg/ml. ISC-CM was further supplemented, as indicated, withfetal calf serum (FCS) (HyClone Laboratories, Logan, Utah) or bovineserum albumin (BSA) (Sigma, St. Louis, Mo.). Phosphate buffered saline,pH 7.2, was supplemented with 3 mM NaN₃ (PBSN).

Example 3 Viruses

PR8 (A/PR/8/34(H1N1)) was a mouse-adapted strain. PR8-SEQ14 was anescape mutant selected from PR8 sequentially in the presence of 14different PR8(HA)-specific monoclonal antibodies. X31 was a reassortantvirus containing all PR8 derived genes except those coding for H3 andN2, which were from (A/Aichi/68(H3N2)) (Kilbourne (1969) Bull. WHO41:643-5). JAP was (A/Japan/305/57(H2N2)) and B/LEE was the type Binfluenza virus strain B/Lee/40.

Example 4 Production of M2e24-Specific Hybridomas

Three M2e24-specific hybridomas (M2-56, M2-80, M2-86) were derived froma BALB/c mouse that had been challenged with two consecutive infections,the first with PR8 and the second with X31. Three days before fusion,the mouse was injected intravenously (i.v.) with 5 μg (4)M2e24-MAA inPBS and spleen cells fused with Sp2/O myeloma cells. Two hybridomas(M2-1, M2-15) were derived from a mouse that had recovered from threeconsecutive heterosubtypic infections (first with PR8, second with X31,third with JAP) and was boosted intranasally (i.n.) with 5 μg(4)M2e24-MAA together with 3 μg phosphorothionated oligodeoxynucleotide(ODN) c1826 and 0.5 μg cholera toxin (CT). Cells from draininglymphnodes (superficial cervical and mediastinal) were fused three dayslater. Hybrid cultures were screened for secretion of antibodies thatreacted in ELISA with (1)M2e24-MAA and/or JAP-infected Madin Darbycanine kidney (MDCK) cells. All M2e24-specific hybridomas generated bythese protocols cross-reacted with M2e24-MAA and JAP-infected MDCK cellsat roughly equimolar amounts, indicating that the M2e-MAAs sharedseveral B cell determinants in common with native M2e. The hybridoma14C2 is well-known in the art (Zebedee and Lamb (1988) supra).

Example 5 Antibody Measurements by ELISA

Wells of Costar serocluster, round-bottom, polyvinyl plates were coatedwith (1)M2e24-MAA by incubation overnight at room temperature (coveredto prevent evaporation) with 25 μL of the construct at 0.5 μg/mL inPBSN. The plates were blocked for one to two hours with PBSN containing1% BSA prior to assay. JAP-MDCK ELISA plates were prepared as follows:MDCK cells were grown to confluency in FALCON®, microtest, flat-bottom,96-well, polystyrene, tc plates, typically by seeding wells with 4×10⁴MDCK cells in 100 μL of ISC-CM containing 5% FCS. After one day ofincubation (37° C., 6% CO₂), monolayers were washed with PBS to removeserum components and infected by incubation (37° C.) with 50 μL ofISC-CM containing ˜10⁶ TCID₅₀ of JAP virus. After one hour, 100 μL ofISC-CM containing 5% FCS was added to each well and incubation continuedas above for six to seven hours. Monolayers were then washed with PBS,fixed by incubation for five minutes at room temperature with 5%buffered FORMALDE-FRESH® (FisherChemical, Pittsburgh, Pa.), washed withPBSN and blocked and stored with PBSN containing 1% BSA at 4° C. InELISA, all test samples and reagents were diluted in PBSN containing 1%BSA, used at 25 μL/round-bottom well or 50 μL/flat-bottom well, andincubated for 90 minutes at room temperature. Bound mouse antibody wasgenerally detected with biotinylated rat-anti-mouse-CK monoclonalantibody 187.1, followed by Streptavidin-AP (Sigma, St. Louis, Mo.) andpNPP (Sigma, St. Louis, Mo.). The pNPP solution was used at 50 μL and100 μL per round- and flat-bottom well, respectively. Absorption wasmeasured with the EMAX® plate reader (Molecular Devices, Sunnyvale,Calif.) and the difference between OD₄₀₅ and OD₇₅₀ (OD₄₀₅₋₇₅₀) recorded,usually after 30-45 minute of incubation. All assays included atitration of a purified monoclonal antibody of appropriate specificityfor quantification of test samples. ELISA data were analyzed with theSOFTMAX PRO® software (Molecular Devices, Sunnyvale, Calif.).

The structurally different M2e24-MAAs showed no significant differencesin reactivity with M2e-specific monoclonal antibodies in ELISA. Allmonoclonal antibodies reacted equally well with equimolar amounts of theMAA containing (4)M2e24-MAA and (4)M2e15-MAP having a proline atposition 10, indicating that the relevant B cell determinant shared bynative and synthetic M2e is formed by the N-terminal region of M2e.

Example 6 Analysis of CD4⁺ T Cell Responses

Antigen presenting cells (APC) were prepared from the spleen of naïveBALB/c mice. PERCOLL™ (PHARMACIA®, Uppsala, Sweden) was added to thecell suspension to give a final concentration of 33%. The suspension wasunderlayed with a small volume of 70% PERCOLL™ and centrifuged (10minutes, 600 grams, room temperature) to remove cell debris anderythrocytes. Cells at the 33%/70% interface were harvested, washed,irradiated (2200 rad) and suspended in ISC-CM at 5×10⁶ cells/mL. Onehundred μL were dispensed per well of flat-bottomed tissue cultureplates. Antigen in ISC-CM was added in 50 μL volumes per well. Fifty μLof responder cell suspension, typically MedLN cells at 10⁷/mL or Thclones at 4×10⁵/mL in ISC-CM, were added per well. One μCi ofH³-thymidine was added during the third (Th clones) or fourth (LNresponder cells) day of incubation. Plates were then frozen and thawedonce and the cells were harvested with a Skatron cell harvester (SkatronInstruments Inc., Sterling, Va.) onto filter mats (Skatron InstrumentsInc., Sterling, Va.). Punched out pieces of filter mat were transferredinto scintillation fluid and counted for radioactivity.

On a molar basis, M2e24-MAAs exhibited similar potency as free S1peptide in stimulating S1-specific Th cells with one notable exception:the mannosylated MAP ((1)M2e24-Man-MAA) exhibited a 100- to 1000-foldhigher stimulatory potency in vitro than free S1 peptide andnon-mannosylated MAAs. The stimulatory potency was comparable topurified intact HA which contains the S1 determinant in the context ofthe intact HA1 polypeptide and also contains mannosylated carbohydrateside chains.

Example 7 Analysis of CD8⁺ Memory T Cell Response

Spleen cells from vaccinated mice were purified as provided in a 33%/70%PERCOLL™ gradient and used as responder cells. A20 cells (H2^(d),positive for MHC class II) were infected with PR8 (10⁶ TCID₅₀/10⁶ A20,one hour at 37° C.), irradiated with 4400 rad, washed and used asstimulators. Cultures (6 mL) were set up in T25 FALCON® flasks andcontained 25×10⁶ responder cells and 10⁶ stimulator cells in ISC-CMcontaining 5% FCS. After five days of incubation (stationary, upright),viable cells were purified in a 33%/70% PERCOLL™ gradient, counted andusing standard methods (Mozdzanowska, et al. (1997) Virology 239:217-25)tested for the ability to induce release of ⁵¹Cr from PR8- andB/LEE-infected P1.HTR target cells during a four-hour incubation period.

Example 8 Immunization Protocols

M2e-MAAs and adjuvants, in a total volume of 50 μL, were placed onto thenares of anesthetized mice (ketamine and xylazine injectedintraperitoneally at 70 mg/kg and 7 mg/kg body weight, respectively),which resulted in its aspiration into the respiratory tract. One dose of50 μL contained 3 μg of M2e-MAA, 3 μg of the ODN 1826 (Krieg, et al.(1995) Nature 374:546-9; Yi, et al. (1998) J. Immunol. 160:4755-61) and0.5 μg of CT (Sigma, St. Louis, Mo.). Adjuvant combination and dosingwas based on standard methods (Mozdzanowska, et al. (1999) supra).Booster inoculations were administered in four to five week intervals.Mice that received adjuvant solution without M2e-MAA were used asnegative controls and mice that had been subjected to two consecutiverespiratory tract infections, first with PR8 and second with PR8-SEQ 14,were used as positive controls.

Example 9 Virus Challenge Experiments

The strength of vaccine-induced protection was tested by i.n. challengeof mice with ˜10³ MID₅₀ (50% mouse infectious dose) of X31. Three dayslater, the mice were anesthetized, exsanguinated by heart puncture, anddissected for collection of nasal, tracheal and pulmonary tissues.Titers of infectious virus were determined by titration of tissuehomogenates in MDCK cell cultures or embryonated hen's eggs usingstandard methodologies (McCluskie and Davis (2000) supra).

Example 10 In Vitro Analysis of Immune Response to MAAs

To induce a Th-dependent antibody response to native viral M2e, M2e-MAAsshared B cell epitopes with native virus-induced M2e and containeddeterminants that could be presented to Th cells. JAP-MDCK cells andM2e-MAAs were compared for their reaction with several M2e-specificmonoclonal antibodies in ELISA. The 14C2 monoclonal antibody wasgenerated from a mouse immunized with purified viral M2 (Zebedee andLamb (1988) supra); all other antibodies were isolated from micerecovered from consecutive influenza type A virus infections and boostedwith (4)M2e24-MAA three days prior to fusion. The final boost with(4)M2e24-MAA was performed to increase the frequency of isolation ofM2e-specific hybridomas. All six M2e-specific monoclonal antibodiesreacted well with both M2e-MAA and JAP-MDCK, though four were slightlymore and two slightly less effective in binding to JAP-MDCK than towells coated with (1)M2e24-MAA at 1.5 ng/well. The data indicated thatM2e-MAAs mimicked effectively several B cell determinants of the nativevirus-induced tetrameric M2e.

The structurally different M2e-MAAs, when used at equimolar M2econcentrations, showed no significant differences in reaction withM2e-specific monoclonal antibodies.

To optimize Th-mediated help, two distinct Th determinants wereincorporated into the MAAs, one (S1) presented by E^(d) and the other(S2) by A^(d). These determinants were identified as the twoimmunodominant targets of the HA(PR8)-specific Th response of BALB/c(H-2^(d)) mice (Gerhard, et al. (1991) J. Virol. 65:364-72). S1corresponds to the HA region 110-120 and S2 to 126-138. However, the S2peptide in the present constructs was altered compared to the native S2by replacing the cysteine at position 135 with serine to avoid formationof disulfide bonds between S2 and the cysteine contained in the M2epeptide.

The efficacy of the MAAs to stimulate S1- and S2-specific Th clones wasdetermined in cultures that contained irradiated BALB/c spleen cells asAPCs, S1- or S2-specific Th clones as responders and variousconcentrations of free S1 or S2 peptides, M2e-MAAs or purified HA.Proliferation of the Th clones was assessed by ³H-thymidineincorporation during the third day of culture. All M2e-MAAs stimulatedthe S1-specific Th clone V2.1 with equal or higher potency than the freeS1 peptide. A 100-fold greater stimulatory potency of the mannosylatedMAA was observed most likely due to improved capture of this MAA bymannose-receptors expressed on APCs (Engering, et al. (1997) Eur. J.Immunol. 27:2417-2; Tan, et al. (1997) Eur. J. Immunol. 27:2426-35). Thestimulatory activity of this MAA is similar, on a molar basis, to theactivity of the HA molecule which also contains mannosylatedcarbohydrate side chains (Keil, et al. (1985) EMBO J. 4:2711-20).

By contrast, none of the MAAs stimulated the S2-specific Th clone5.1-5R6. This Th clone responded well to stimulation with the isolatednative S2 peptide and intact HA, thus the change of Cys(135) to Ser mayhave reduced its stimulatory potency for this Th clone. Two additional,clonally unrelated, S2-specific Th clones were tested and also failed torespond to MAAs. Since the Cys(135)→Ser does not to affect the peptide'sability to bind to A^(d) (Sette, et al. (1989) J. Immunol. 142:35-40),it may form an antigenically novel Th determinant which is notrecognized by Th specific for the native S2 determinant. Crystalstructure analysis of the S2/A^(d) complex indicated that the amino acidat position 135 is not an anchor residue (Scott, et al. (1998) Immunity8:319-29).

Thus, the in vitro analyses indicated that the M2e-MAAs mimicked B celldeterminants of the native virus-induced M2e and contained at least onefunctional Th determinant.

1. A multiple antigenic agent comprising:

(SEQ ID NO:1) wherein, R₁ is absent or a nucleic acid sequence; m is atleast 1; n is at least 1; Xaa₁ is 0 to 1 amino acid residue comprising

or Gly; R₂, R₃ and R₄ are independently a B cell determinant, a T celldeterminant, or targeting molecule; and R₅ is alanine.
 2. The multipleantigenic agent of claim 1, wherein when m is greater than 1, each R₂can independently be a B cell determinant, a T cell determinant, or atargeting molecule; and when n is greater than 1, each R₃ canindependently be a B cell determinant, a T cell determinant, or atargeting molecule.
 3. The multiple antigenic agent of claim 1, whereinthe B cell determinant comprises the ectodomain of influenza matrixprotein 2 or a homolog thereof.
 4. A composition comprising the multipleantigenic agent of claim 1 and a pharmaceutically acceptable carrier. 5.The composition of claim 4, wherein the composition further comprises anadjuvant.
 6. The composition of claim 4, wherein the compositioncomprises a vaccine.
 7. The composition of claim 5, wherein thecomposition comprises a vaccine.
 8. A method for preventing or treatinga viral infection comprising administering to a subject an effectiveamount of a composition of claim 4 to prevent or treat the signs orsymptoms of a viral infection.
 9. A method for preventing or treating aviral infection comprising administering to a subject an effectiveamount of a composition of claim 5 to prevent or treat the signs orsymptoms of a viral infection.
 10. A method for preventing or treating aviral infection comprising administering to a subject an effectiveamount of a composition of claim 6 to prevent or treat the signs orsymptoms of a viral infection.
 11. A method for preventing or treating aviral infection comprising administering to a subject an effectiveamount of a composition of claim 7 to prevent or treat the signs orsymptoms of a viral infection.
 12. The method of claim 8, wherein theviral infection comprises influenza type A virus.
 13. The method ofclaim 9, wherein the viral infection comprises influenza type A virus.14. The method of claim 10, wherein the viral infection comprisesinfluenza type A virus.
 15. The method of claim 11, wherein the viralinfection comprises influenza type A virus.